5310
J. Med. Chem. 2008, 51, 5310–5319
Cytotoxicity, Cellular Uptake, and DNA Interactions of New Monodentate Ruthenium(II) Complexes Containing Terphenyl Arenes Tijana Bugarcic,†,‡,⊥ Olga Nova´kova´,§,⊥ Anna Hala´mikova´,§ Lenka Zerza´nkova´,§ Oldrˇich Vra´na,§ Jana Kasˇpa´rkova´,§,| Abraha Habtemariam,‡ Simon Parsons,† Peter J. Sadler,‡ and Viktor Brabec*,§ School of Chemistry, UniVersity of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, United Kingdom, Department of Chemistry, UniVersity of Warwick, Gibbet Hill Road, CV4 7AL, United Kingdom, Institute of Biophysics, Academy of Sciences of the Czech Republic, V.V.i., KraloVopolska 135, CZ-61265 Brno, Czech Republic, Laboratory of Biophysics, Department of Experimental Physics, Faculty of Sciences, Palacky UniVersity, tr. SVobody 26, CZ-771 46 Olomouc, Czech Republic ReceiVed March 19, 2008
We have compared the cancer cell cytotoxicity, cell uptake, and DNA binding properties of the isomeric terphenyl complexes [(η6-arene)Ru(en)Cl]+, where the arene is ortho- (2), meta- (3), or para-terphenyl (1) (o-, m-, or p-terp). Complex 1, the X-ray crystal structure of which confirms that it has the classical “pianostool” geometry, has a similar potency to cisplatin but is not cross-resistant and has a much higher activity than 2 or 3. The extent of Ru uptake into A2780 or A2780cis cells does not correlate with potency. Complex 1 binds to DNA rapidly and quantitatively, preferentially to guanine residues, and causes significant DNA unwinding. Circular and linear dichroism, competitive binding experiments with ethidium bromide, DNA melting, and surface-enhanced Raman spectroscopic data are consistent with combined intercalative and monofunctional (coordination) binding mode of complex 1. This unusual DNA binding mode may therefore make a major contribution to the high potency of complex 1. Introduction Organoruthenium complexes of the type [(η6-arene)RuII(en)Cl]+, where arene ) benzene or a benzene derivative and en ) 1,2diaminoethane, exhibit anticancer activity, including activity against cisplatin (cis-diamminedichloridoplatinum(II)) resistant cancer cells.1,2 This class of complexes can form strong monofunctional adducts with DNA.2 Modifications of natural DNA by [(η6-bip)Ru(en)Cl]+, where bip ) biphenyla, studied using several different techniques,3 have shown preferential binding to guanine (G) residues. [(η6-bip)Ru(en)Cl]+ binds to DNA through coordination to G N7 as well as noncovalently, through hydrophobic interactions between the arene and DNA. These hydrophobic interactions may include intercalation of the noncoordinated phenyl ring between DNA bases and minor groove binding. Intramolecular π-π arene-nucleobase stacking has been observed in the crystal structure of [(η6-bip)Ru(en)(9EtG-N7)][PF6]2,4 and strong H-bonding between an NH of en and C6O from G contributes to the G specificity.5 We have synthesized new complexes of the type [(η6arene)Ru(en)Cl]+, where the arene is ortho-, meta-, or para* To whom correspondence should be addressed. Phone: +420541517148. Fax: +420-541240499. E-mail:
[email protected]. † University of Edinburgh. ‡ University of Warwick. § Institute of Biophysics, Brno. | Palacky University, Olomouc. ⊥ The first two authors are joint first authors. a Abbreviations: bip, biphenyl; bp, base pair; cisplatin, cis-diamminedichloridoplatinum(II); CD, circular dichroism; CT, calf thymus; dien, bis(2-aminoethyl)amine; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; EtBr, ethidium bromide; en, 1,2-diaminoethane; FAAS, flameless atomic absorption spectrophotometry; IC50, concentration inhibiting cell growth by 50%; ICD, induced circular dichroism; LD, linear dichroism; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; rb, the number of atoms of the metal bound per nucleotide residue; ri, the molar ratio of free metal complex to nucleotide-phosphates at the onset of incubation with DNA; SERS, surface-enhanced Raman spectrometry; t50%, the times at which the binding reached 50%; terp, terphenyl; tm, DNA melting temperature; ∆tm, the difference between the tm values of ruthenated and nonmodified DNAs.
Figure 1. Structures of RuII arene complexes. 1, [(η6-p-terp)Ru(en)Cl]+; 2, [(η6-o-terp)Ru(en)Cl]+; 3, [(η6-m-terp)Ru(en)Cl]+.
terphenyl (o-, m-, or p-terp; Figure 1) to investigate the effect on cytotoxicity of an additional phenyl ring compared to bip as arene. Such complexes are expected to show enhanced arene intercalation compared to the bip analogue. In the case of p-terp, Ru is bound to a terminal phenyl ring, whereas in the cases of o- and m-terp, Ru is bound to the central phenyl ring. Aromatic hydrocarbons consisting of a chain of three benzene rings, terphenyls (terps), have three isomers in which the terminal rings are o-, m-, or p-substituents of the central ring. Most of the natural terphenyls are p-terp derivatives. Very few m-terp derivatives occur naturally, and o-terps have not been found in nature.6 In recent years, it has been reported that some terphenyls exhibit significant biological activity such as neuroprotective, antithrombotic, anticoagulant, and cytotoxic activity. It has also been found that some popular edible mushrooms are rich in terphenyls, a sign that the toxicity of terphenyls is low. We have investigated in the present work the relationship between cytotoxicity, cell uptake of ruthenium, and DNA binding for the group of complexes consisting of the p-, o-, and m-terp arenes (complexes 1, 2, and 3, respectively). Their activity toward human ovarian tumor cell lines A2780 (parent, cisplatin-sensitive) and A2780cisR (with acquired cisplatin resistance), human ovarian carcinoma CH1 (cisplatin sensitive),
10.1021/jm8003043 CCC: $40.75 2008 American Chemical Society Published on Web 08/14/2008
Monodentate Ru(II) Complexes with Terphenyl Arenes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5311 Table 3. IC50 Mean Values (µM) Obtained for RuII Arene Complexes Tested in the Present Worka,b c
complex
CH1
SKBR3
A2780
A2780cisR
cisplatin 1 2 3
0.9 ( 0.1 2.2 ( 0.3 23 ( 1 51 ( 9
8(2 8(1 13 ( 3 80 ( 5
2.8 ( 0.7 4(1 30 ( 4 42 ( 4
18.6 ( 0.4 (6.7) 1.4 ( 0.6 (0.4) 18.3 ( 0.8 (0.6) 31 ( 4 (0.7)
a Drug treatment period was 72 h. b The experiments were performed in quadruplicate. c Resistance factor, defined as IC50 (resistant)/IC50 (sensitive), is given in parentheses.
Figure 2. ORTEP diagram for cation of complex 1 [(η6-p-terp)Ru(en)Cl][PF6], with 50% probability thermal ellipsoids. All hydrogen atoms have been omitted for clarity. Table 1. X-ray Crystal Structure Data for Complex 1 formula
C20H22ClF6N2PRu
molar mass crystal system crystal size/mm space group crystal a/Å b/Å c/Å R/deg β/deg γ/deg T/K Z R [F > 4σ (F)]a Rwb GOFc ∆F max and min/eÅ-3
571.89 monoclinic 0.27 × 0.21 × 0.21 P21/c yellow/block 19.0359(10) 10.1405(5) 11.2966(5) 90 105.218(3) 90 150(2) 4 0.0479 0.1256 1.041 1.557, -0.814
a R ) ∑||Fo| - |Fc||/∑|Fo|. b Rw ) [∑w(Fo2 - Fc2)2/∑wFo2)]1/2. c GOF ) [∑w(Fo2 - Fc2)2/(n - p)]1/2, where n ) number of reflections and p ) number of parameters.
Table 2. Selected Bond Lengths (Å) and Angles (deg) for Complex 1 bond/angle
length/angle
Ru-Cl Ru-N12 Ru-N42 Ru-C11 Ru-C21 Ru-C31 Ru-C41 Ru-C51 Ru-C61 N12-Ru-N42 N12-Ru-Cl N42-Ru-Cl
2.3929 (11) 2.125 (4) 2.126 (4) 2.174 (4) 2.152 (5) 2.190 (4) 2.225 (4) 2.191 (4) 2.157 (5) 78.76 (14) 83.65 (10) 84.57 (11)
and human mammary carcinoma SKBR3 (intrinsically cisplatin resistant) cell lines was also investigated. Results Crystal Structure. The X-ray crystal structure of the cation of complex 1 ([(η6-p-terp)Ru(en)Cl][PF6]) is shown in Figure 2. The crystallographic data are listed in Table 1, and selected bond lengths and angles in Table 2. In the complex, RuII adopts the familiar “three-legged piano-stool” geometry with an η6 π-bonded arene, forming the seat of the stool. The legs of the stool are Cl and the N atoms of the en chelating ligand. The Ru-Cl bond length in complex 1 is 2.3929(11) Å (Table 2). A twisting of the phenyl rings is present in the crystal structure of complex 1. The twist angle between the bound and the central ring is 43.21°, and between the central and the terminal ring 38.47°. The planes of the terminal and bound rings are twisted
Table 4. Uptake of RuII Arene Complexes and Cisplatin into Cellsa,b A2780 A2780cisR
cisplatin
complex 1
complex 2
complex 3
30 ( 2 47 ( 3
350 ( 20 150 ( 10
2130 ( 130 1010 ( 90
630 ( 50 490 ( 80
a Table shows the uptake of cisplatin and complexes 1-3 at their equitoxic concentrations (corresponding to the IC50 values shown in Table 1) into A2780 or A2780cisR cells after 72 h. Each value shown in the Table 4 is in pmole of Ru or Pt/106 cells. b The experiments were performed in triplicate.
by 5.62°. A CH-π interaction is present between the C61H proton of the bound ring of p-terp (Figure S1 of Supporting Information) from one molecule and the center of the π-system of the central ring of p-terp from another molecule (distance ) 2.624 Å). The distances between carbons of the bound arene ring and the metal center range from 2.152(5) to 2.225(4) Å. Cytotoxicity. The cytotoxic activity of the new RuII arene complexes 1-3 was determined against four different cisplatin sensitive and resistant cancer cell lines (Table 3). The human ovarian carcinoma cell lines A2780, CH1 (both cisplatin sensitive), A2780cisR (with acquired cisplatin resistance), and human mammary carcinoma cell line SKBR3 (intrinsically cisplatin resistant) were employed. A2780cisR cells are resistant to cisplatin through a combination of decreased uptake, enhanced DNA repair/tolerance, and elevated reduced-glutathione levels.7,8 The tumor cell lines were incubated for 72 h with RuII arene complexes or cisplatin, and the cell survival in the culture treated with RuII complexes was evaluated as described in the Experimental Section. All complexes show activity and their corresponding IC50 values (IC50 ) concentration inhibiting cell growth by 50%) are reported in Table 3. In general, activity follows the order 1, cisplatin . 2 > 3, with all Ru complexes lacking cross-resistance with cisplatin. Cellular Ruthenium Complex Uptake. An important factor that usually contributes to transition metal-based drug cytotoxicity is cellular uptake. To examine accumulation of complexes 1-3, the cellular levels of these RuII arene complexes were measured after a 72 h exposure of the A2780 and A2780cisR cells to the drugs at equitoxic concentrations (i.e., at the concentrations corresponding to the IC50 values shown in Table 3). The amount of ruthenium in cells (in pmol/106 cells, Table 4), 2 . 3 > 1, does not correlate with their cytotoxicity (IC50 value). Because complexes 1-3 were tested at their equitoxic doses, the results shown in Table 4 imply that notably less molecules of complex 1 inside the cells in comparison with complexes 2 and 3 are necessary to induce the same cytotoxic effect. DNA-Bound Ruthenium in Cells Exposed to RuII Arene Complexes. Distortions of DNA structure often correlate with anticancer activity.9,10 Hence, it is of great importance to understand in detail DNA binding properties of the new RuII arene complexes and their possible relationship to cytotoxicity in tumor cell lines. Complexes 1 and 3 exhibited the highest and lowest potency, respectively (Table 1), so these complexes were selected for a more detailed DNA binding study. We
5312 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Bugarcic et al.
Figure 3. Kinetics of the binding of complexes 1 (9) and 3 (4) to calf thymus DNA in the medium of 10 mM NaClO4, at 37 °C and pH 6. The concentration of DNA was 3 × 10-4 M (related to the monomeric nucleotide content), and ri was 0.08.
examined DNA-bound ruthenium in A2780 cells after exposure to complexes 1 and 3. Measurements of DNA-bound ruthenium after 2 h of 50 µM drug exposure revealed that the amount of ruthenation by complexes 1 and 3 was 5.0 ( 2.0 and 14.0 ( 4.0 pg Ru/µg DNA, respectively. Hence, complex 1 requires fewer DNA lesions than does complex 3 to achieve cell growth inhibition. DNA Binding in Cell-Free Media. Kinetics of Binding to Calf Thymus (CT) DNA. The rate of binding of RuII arene complexes 1 and 3 to CT DNA was determined at an ri (molar ratio of free RuII arene complex to nucleotide phosphate) ratio of 0.08 in 10 mM NaClO4 at 37 °C in the dark. RuII arene complexes were incubated with the CT DNA and aliquots removed at various time intervals, rapidly cooled, precipitated out by addition of ethanol, and the content of the supernatant determined by flameless atomic absorption spectrometry (FAAS) (Figure 3). Intriguingly, both complexes bind rapidly (t50% ca. 5 and 9 min for complexes 1 and 3, respectively). Complex 1 binds almost quantitatively, whereas only ca. 80% of the complex 3 is bound after 48 h. The binding experiments carried out in this work indicated that modification reactions resulted in the irreversible coordination of the RuII arene complexes 1 and 3 to CT DNA, which thus facilitated sample analysis. Hence, it was possible to prepare samples of DNA modified by complexes 1 or 3 at a preselected value of rb (rb values are defined as the number of atoms of the metal bound per nucleotide residue). Thus, except where stated, samples of DNA modified by RuII arene compounds 1 and 3 and analyzed further by biophysical or biochemical methods were prepared in 10 mM NaClO4 at 37 °C. After 24 h of the reaction of DNA with the complex, the samples were precipitated in ethanol and dissolved in the medium necessary for a particular analysis, and the rb value in an aliquot of this sample was checked by FAAS. In this way, all analyses described in the present paper were performed in the absence of unbound (free) RuII arene complex. Transcription Mapping. Cutting of pSP73KB DNA by NdeI and HpaI restriction endonucleases yielded a 212-base pairs (bp) fragment (a substantial part of its nucleotide sequence is shown in Figure 4B). This fragment contained T7 RNA polymerase promotor. In vitro RNA synthesis by RNA polymerases on this DNA template modified by RuII arene complexes 1 and 3 at the same level of the ruthenation (rb ) 0.01) can be prematurely terminated at the level or in the proximity of adducts (Figure 4A). Interestingly, monofunctional DNA adducts of several platinum complexes are unable to terminate RNA synthesis.11–13 The major stop sites, primarily guanine residues, were roughly identical for both RuII arene complexes (Figure 4B). The profiles are similar to that obtained for DNA treated with the anticancer drug cisplatin (lane cisPt in Figure 4A) and also to those reported
Figure 4. Inhibition of RNA synthesis by T7 RNA polymerase on the NdeI/HpaI fragment of pSP73KB plasmid modified by RuII arene complexes and cisplatin. (A) Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of RNA synthesis by T7 RNA polymerase on the NdeI/HpaI fragment containing adducts of RuII arene complexes and cisplatin. Lanes: control, unmodified template; A, U, G and C, chain terminated marker DNAs; cisPt, 1 and 3, the template modified by cisPt, RuII arene complexes 1 or 3 at rb ) 0.01, respectively. (B) Schematic diagram showing the portion of the sequence used to monitor inhibition of RNA synthesis by cisplatin and RuII arene complexes. The arrow indicates the start of the T7 RNA polymerase, which used as template the upper strand of the NdeI/HpaI fragment of pSP73KB. The open and closed bullets represent major stop signals for DNA modified by cisplatin or complex 1, respectively. The numbers correspond to the nucleotide numbering in the sequence map of the pSP73KB plasmid.
previously for other type of RuII arene complexes such as [(η6arene)Ru(en)Cl]+, where arene ) biphenyl, dihydroanthracene, tetrahydroanthracene, p-cymene, or benzene.3 The major stop sites for DNA modified by complex 1 and cisplatin are demonstrated in Figure 4B. Thus, these results suggest that the major sites in DNA at which complexes 1 and 3 preferentially bind are guanine residues. Circular Dichroism (CD). To gain further information, we also recorded CD spectra of DNA modified by complexes 1 and 3 (Figure 5). Complexes 1 and 3 have no intrinsic CD signals, as they are achiral so that any CD signal above 300 nm can be attributed to the interaction of complexes with DNA. Below 300 nm, any change from the DNA spectrum is due either to the DNA induced CD (ICD) of the metal complex or the metal complex induced perturbation of the DNA spectrum. The signature of complex 1 bound to CT DNA is a strong negative ICD at around 308 nm and a strong positive ICD centered at 376 nm, with the crossover at 351 nm (Figure 5A). On the other hand, the signature of complex 3 bound to CT DNA is only a very weak and broad positive ICD centered at 376 nm (Figure 5B). These results reflect different binding modes of complexes 1 and 3 to DNA. Unfortunately these complexes also absorb in the DNA region (Figure 5C) so that this ICD signal is due to changes in both the intrinsic DNA CD and the ligand-induced
Monodentate Ru(II) Complexes with Terphenyl Arenes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5313
Figure 5. Circular dichroism (CD) spectra (A,B) of calf thymus DNA (1 × 10-4 M) modified by complexes 1 and 3 and UV-vis spectra of complexes 1 and 3 (C); the medium was 10 mM NaClO4, pH 6. (A) DNA was modified by complex 1 at rb ) 0, 0.013, 0.029, 0.087, 0.125 (curves 1-5, respectively). (B) DNA was modified by complex 3 at rb ) 0, 0.013, 0.033, 0.071, 0.118 (curves 1-5, respectively). (C) Complexes 1 and 3 were at the concentration of 2.7 × 10-5 and 2.75 × 10-5 M, respectively. The arrows in (A,B) show a change of CD with increasing rb value.
Figure 7. Plots of the EtBr fluorescence versus rb for DNA modified by cisplatin, [PtCl(dien)]Cl, and RuII arene complexes in 10 mM NaClO4 at 37 °C for 24 h: (×), [PtCl(dien)]Cl; (9), complex 1; (0), complex 3. Data points measured in triplicate varied on average ( 3% from their mean. Figure 6. Linear dichroism (LD) spectra of calf thymus DNA modified by RuII arene complexes. LD spectra were recorded for DNA in 10 mM NaClO4, at pH 6.0. The concentration of DNA was 3 × 10-4 M and rb was 0.1. Thick solid line: control, nonmodified DNA. Dotted line: DNA modified by complex 1. Dashed line: DNA modified by complex 3.
CD, which impedes unambiguous interpretation of the CD spectra in Figure 5A,B in the DNA region (
